The invention relates to a method for in vitro diagnosis of endometriosis.
Endometriosis is one of the most frequently occurring gynecological diseases, by which roughly 5-10% of all women of child-bearing age are affected (Sillem, M. 1998; Programmed(R) 23, Suppl. 1, 1-28). It is characterized by the occurrence of endometrial tissue outside of the physiological mucous membrane lining of the uterus. In addition to pain and numerous other symptoms, many endometriosis patients are sterile, and a large portion of IVF patients (IVF=in vitro fertilization) suffer from endometriosis (Adamson, G. D. 1997; Sem. Reprod. Biol. 15, 263-271). Very recently, publications that speak for a genetic predisposition in the development of an endometriosis have been multiplying (Kennedy, S. 1997; Sem. Reprod. Biol. 15, 309-318). The loss of tumor suppressor molecules and family clusters in the case of endometriosis patients was thus described.
Endometriosis is currently diagnosed with the aid of laparoscopy. This is an invasive method, which frequently results in complications (Chapron, C. et al. 1998; Hum. Reprod. 13, 867-872; Jansen, F. W. et al., 1997; Br. J. Obstet. Gynecol. 104, 595-600). It is performed under anesthesia and requires a fully set-up operating room.
There is therefore a need for new diagnostic methods. A method that would impose less of a burden for the patients and that could be performed by the attending physician would be desirable.
This problem is achieved according to the invention by the identification of genes that are differentially regulated in endometriosis and the preparation of a method for detection of their gene products.
The invention relates to a method for in vitro diagnosis of endometriosis, whereby the amount of gene product of at least one gene from the group that consists of fibronectin, insulin-like growth factor binding protein-2, transmembrane receptor PTK7, platelet-derived growth factor receptor alpha, collagen type XVIII alpha 1, subtilisin-like protein (PACE4), laminin M chain (merosin), elastin, collagen type IV alpha 2, p27 interferon alpha-inducible gene, reticulocalbin, aldehyde dehydrogenase 6, gravin, nidogen and phospholipase C epsilon is determined in a patient sample and is compared to the amount of this gene product in a control sample, whereby a smaller amount of this gene product indicates the presence of an endometriosis.
The group of genes is described in more detail in FIG. 1. The expression strength, i.e., the amount of gene product, is determined by at least one of the genes in a patient sample that is mentioned in FIGS. 1a and 1b and compared to that of a control sample (women without endometriosis). The samples that are to be compared must both originate from the secretory phase, thus from the range of days 15-28 after the last menstruation. A decreased expression strength of at least one of the above-mentioned genes in the patient sample indicates the presence of an endometriosis.
A patient sample can be a sample from endometrial tissue, peritoneal fluid, blood, vaginal secretion or urine of the patient.
A gene product is either mRNA, the cDNA that is derived therefrom, a polypeptide or portions of a polypeptide. The amino acid sequences of the polypeptides are depicted in FIGS. 2a-m. 
The methods according to the invention can be used for first-time diagnosis of endometriosis. In this case, the amount of the gene product in the patient sample is compared to a control sample of undiseased women. The method according to the invention can also be used to evaluate the course of the disease. Thus, e.g., the success of a therapy can be determined. In this case, the patient sample is compared to a prior sample from the same patient.
The gene product polypeptide or a segment of a polypeptide is detected by immunoassay. To this end, specific antibodies are produced using one or more polypeptides that are selected from the group that is described in FIGS. 2a-m. The antibodies can be monoclonal or polyclonal. They can be directed against respectively the entire polypeptide or against fragments thereof. Such an antibody is obtained according to standard methods by immunization of test animals. The antibodies are then used in, e.g., an ELISA (enzyme-linked-immunosorbent assay), in an RIA (radioimmunoassay) or in the immunohistochemistry for determining the amount of the gene product (Aoki, K. et al. 1996; Forensic Sci. Int. 80, 163-173).
The invention further relates to the use of an antibody chip according to the invention for diagnosis of endometriosis. Antibody chips are miniaturized vehicles, in most cases made of glass or silicon, on whose surface antibodies of known specificity are immobilized in an ordered grid of high density. The detection of the protein/protein interactions can be carried out by mass spectrometry, fluorescence or surface plasmon resonance. Antibodies that specifically bind the proteins that are selected from the group that is described in FIGS. 2a-m can be immobilized on the antibody chip. Methods for the production and use of antibody chips are described in Kreider BL, Med Res Rev 2000, 20:212-215.
The mRNA gene products or the cDNA derived therefrom can be determined by hybridization with oligonucleotides, e.g., by a Northern Blot. These oligonucleotides have sequences that are complementary to partial sequences of the gene product that is to be detected and can be labeled with, e.g., a chromogenic, radioactive or fluorescent group. Before hybridization, the cDNA can be amplified with the aid of PCR (Sambrook, J. et al. 1989; Cold Spring Harbor Laboratory Press).
The mRNA gene products or the cDNA derived therefrom can also be determined by quantitative PCR (polymerase chain reaction).
The mRNA can also be determined by in situ hybridization with antisense-RNA. In this case, the antisense-RNA can be labeled with dioxigenin, 32P or 33P. Antisense nucleic acid is a DNA and/or RNA, which is complementary to an mRNA. It can comprise the entire complementary sequence or partial sequences. This method is known to one skilled in the art (Barlati, S. et al. 1999; Histol. Histopathol. 14, 1231-1240).
The hybridization can also be carried out with the aid of a DNA chip. In addition, the invention therefore relates to a DNA chip, on which at least one oligonucleotide is immobilized, which corresponds to the complete cDNA sequence or a partial sequence or a complementary sequence of a gene that is selected from the group that is described in FIGS. 1a and 1b. The invention thus further relates to the use of a DNA chip according to the invention for diagnosis of endometriosis.
DNA chips, also known as DNA microarrays, are miniaturized vehicles, in most cases made of glass or silicon, on whose surface DNA molecules of known sequence are immobilized in an ordered grid of high density. The surface-bonded DNA molecules are hybridized with complementary, optionally labeled nucleic acids. The labeling can be a fluorescence dye.
In the case of oligonucleotide chips, the oligonucleotides that can be bonded to a DNA chip according to the invention represent partial sequences of the gene products (mRNA or cDNA derived therefrom) in the sense or antisense direction. One or more oligonucleotides per gene can be bonded to the DNA chip. Preferred are 25 nucleotide-long oligonucleotides, which are derived from the non-coding strand. The latter are preferably selected from the respective 3xe2x80x2-untranslated end of the gene. For detection, oligonucleotides of one gene, several genes or all genes that are selected from the group that is described in FIG. 1 can be used. Methods for the production and use of DNA chips are described in, e.g., U.S. Pat. Nos. 5,578,832, 5,556,752 and 5,510,270.
In the case of cDNA chips, the complete gene products (cDNAs) or subfragments (200-500 bp long) are bonded to the chip. The method is described in, e.g., Eckmann, L. et al., J. Biol. Chem. 2000, 275: 14084-14094.
The mRNA gene product can also be determined by chromogenic assays.